This study will address mechanisms of substrate recognition by ubiquitin ligases and functions of ubiquitin modifications in transcriptional regulation. Ubiquitination of the yeast Mat?2 transcriptional repressor (?2) is necessary for its Cdc48-dependent removal from DNA as well as its degradation by the proteasome. The attachment of ubiquitin to ?2 is mediated by two distinct pathways, one of which involves the heterodimeric E3 ubiquitin ligase Slx57Slx8. My hypothesis is that a specific region or regions of the ?2 protein is/are required for its recognition by Slx57Slx8. Therefore, this proposal aims to identify a degradation signal (degron) in ?2 that is recognized by Slx57Slx8. The main experimental approach that will be employed for this project is random mutagenesis of the MAT??2 gene to generate single amino acid changes in the ?2 protein that impair its recognition by Slx57Slx8. The recognition of substrates by Slx57Slx8 is of great interest because while ?2 is one of only two confirmed endogenous substrates for Slx57Slx8, accumulating evidence implicates Slx57Slx8 in the degradation of SUMOylated proteins (or proteins with SUMO-like domains) to ensure proper DNA damage repair and cell cycle progression. Slx57Slx8 is a member of the evolutionarily conserved SUMO-targeted ubiquitin ligase (STUbL) family of E3 ubiquitin ligases that includes the human protein RNF4. Thus, the mechanisms elucidated during this project will offer insight into other ubiquitin ligases and other cellular processes. This proposal will also investigate a mutant of ?2 (?2 -3A) that has reduced affinity for DNA and a longer half-life than wild-type ?2. The aim of this project is to determine the effect of DNA binding on the ubiquitin-mediated degradation of ?2. This project will compare wild-type ?2 to ?2 -3A in assays of genetic interaction, protein.DNA interaction, and protein.protein interaction. My main hypothesis is that 12-3A is a poor substrate for the proteasome because it fails to properly interact with the AAA ATPase Cdc48 and/or Cdc48 cofactors. This hypothesis includes the postulate that the activity of Cdc48 (and cofactors) on ?2 is restricted to DNA-associated 12. While the exact functions of Cdc48 and its cofactors in chromatin remodeling are unclear, it is probable that these factors have the ability to alter the composition of several DNA-associated protein complexes in a ubiquitin-dependent manner. Since many transcriptional regulators and other DNA-binding proteins are known to be ubiquitinated, this project is likely to have implications beyond the ?2 system. PUBLIC HEALTH RELEVANCE: The proper growth and function of human cells requires the timely attachment and removal of a small protein called ubiquitin to and from other proteins. Defects in the enzymes that control ubiquitin attachment or removal are known to cause human developmental abnormalities, neurodegenerative disorders, and many different forms of cancer. This project aims to strengthen our understanding of the enzymes that attach ubiquitin to other proteins, with the long-term goal of developing therapies to treat cancers, neurological disorders, and other human diseases.